Neurological dysfunction is a devastating complication of HIV disease. Although potent antiretroviral therapy can control infection with HIV, a long-lived reservoir of virus remains in cells in the brain. This viral reservoir represents a serious impediment to the goal of eradicating HIV, and emphasizes the need for a fundamental understanding of the mechanisms of virus entry and persistence that underlie neurotropism and neurovirulence. Studies performed under the original award showed that CCR5 is the principal coreceptor HIV uses to enter microglia, whereas a subset of strains can use CXCR4 for entry. We also showed that three primary R5 viruses isolated from the brain of two patients who had HIV encephalitis (HIVE) and HIV-associated dementia (HAD) had reduced CCR5/CD4 dependence. We analyzed one of these R5 viruses in detail and found that it had increased CCR5 affinity and, when compared to other primary R5 viruses, increased resistance to CCR5 small molecule inhibitors. The goal of this proposal is to perform a detailed characterization of the role of CCR5 and CXCR4 in virus entry and pathogenesis in the CNS using viruses and tissue samples from patients with HIVE and HAD. The specific aims are: 1) To determine whether HIV viruses that have increased affinity for CCR5 and/or reduced dependence on CCR5/CD4 are more frequent in brain compared to lymphoid tissues, and whether viruses with these properties are associated with HIVE and HAD; 2) To define molecular determinants in the H1V Env that underlie virus tropism for microglia, and determine the influence of these regions of the Env on coreceptor affinity, dependence on CCR5/CXCR4/CD4 levels, and exposure of the coreceptor binding site; and 3) To determine whether viruses that have increased resistance to CCR5 inhibitors are more frequent in brain compared to lymphoid tissues and whether HIV escape mutants that become resistant to CCR5 inhibitors and require lower levels of CCR5 for entry have increased tropism for microglia. The studies will provide insights that are fundamental to understanding HIV pathogenesis in the CNS and will elucidate the potential of chemokine receptors as targets for therapeutic intervention.